Inhibition of nerve cell death by inhibiting degradation of shc3, atf6 or crebl1 by htra2 and method of ameliorating neurodegenerative diseases

ABSTRACT

A novel protein involved in cell-death and a gene encoding the protein, the use thereof to screen for an anti-cell-death factor for use in the treatment of a disorder caused by excessive cell-death or to screen for a remedy for a disorder caused by aberrant suppression of cell-death, and the screening method are provided. Human HtrA2 and human HtrA2-expressing cells and animals to screen for an anti-cell-death factor for use in the treatment of a disorder caused by excessive cell-death, a method for screening for an anti-cell-death factor for use in the treatment of disorder caused by excessive cell-death, and a method for screening for a remedy for a disorder caused by aberrant suppression of cell-death.

TECHNICAL FIELD

The present invention relates to inhibition of the degradation by HtrA2of at least one of SHC3 (src homology 2 domain containing transformingprotein C3), ATF6 (activating transcription factor 6), and CREBL1 (cAMPresponsive element binding protein-like 1). More concretely, the presentinvention relates to a method for inhibiting neural cell death,comprising inhibiting the degradation by HtrA2 of at least one of SHC3,ATF6 and CREBL1, or a method for inhibiting neural cell death,comprising using one or more compounds that inhibit the degradation.Further, the present invention relates to a method for preventing,treating or controlling brain ischemia or neurodegenerative disease,comprising inhibiting the degradation by HtrA2 of at least one of SHC3,ATF6 and CREBL1. Still further, the present invention relates to amethod of identifying a compound that inhibits the degradation by HtrA2of at least one of SHC3, ATF6 and CREBL1. Further, the present inventionrelates to an agent for inhibiting neural cell death, comprising one ormore compounds that inhibit the degradation by HtrA2 of at least one ofSHC3, ATF6 and CREBL1. Still further, the present invention relates toan agent for preventing, treating or controlling brain ischemia orneurodegenerative disease, comprising one or more compounds that inhibitthe degradation by HtrA2 of at least one of SHC3, ATF6 and CREBL1.Further, the present invention relates to a reagent kit, comprising atleast one selected from the group consisting of HtrA2, a polynucleotideencoding HtrA2, and a vector containing the polynucleotide encodingHtrA2; and at least one selected from the group consisting of SHC3,ATF6, CREBL1, a polynucleotide encoding at least one of SHC3, ATF6 andCREBL1, and a vector containing the polynucleotide encoding at least oneof SHC3, ATF6 and CREBL1.

BACKGROUND ART

Organisms are taking self defense against stresses formed on theenvironmentals, by regulating the survival and apoptosis (cell death) ofcells, but the disturbed regulation of the survival andapoptosis-controlling mechanism may cause excessive cell death to inducevarious disorders (Non-Patent Reference 1).

In recent years, researches have been remarkable progressed onmitochondria and endoplasmic reticulum as sites for perceiving andregulating stresses. It has been reported that HtrA2 protein(hereinafter referred to as HtrA2) located at a mitochondrial membranetranslocates from the mitochondrial membrane to cytoplasm due to stresssuch as UV and heat shock (Non-Patent References 2-5), thereby inducestwo types of cell death, that is, caspase dependent cell death andcaspase independent cell death (Non-Patent Reference 6). HtrA2 precursorprotein (SEQ ID NO: 2) converts to the mature type (SEQ ID NO: 4) bybeing cleaved the N-terminal 133 amino acids thereof and thentranslocates from mitochondria to cytoplasm. The mature HtrA2 has aprotease activity. Herein after, HtrA2 with the protease activity may bereferred to as active HtrA2.

It is known that caspase independent cell death is depending on thefeature of HtrA2 itself as a senne protease and that caspase independentcell death is not triggered by the HtrA2 mutant with 306^(th) serine inthe amino acid sequence of the precursor protein thereof (this positionis corresponding to 174^(th) in mature type) substituted with alanine(Non-Patent References 3-5). Such HtrA2 mutant has no protease activity.Herein after, HtrA2 without the protease activity may be referred to asinactive HtrA2. As mentioned, HtrA2 is a factor that plays importantroles in the mechanism of inducing caspase independent cell death.

The References cited in the specification are listed as follows:

Non-patent Reference 1: “Saibou Kougaku (Cell Technology)”, 2002,Vol.21, No.4, p.360-394.

Non-patent Reference 2: Yamaguchi H. et al., “Cancer Research”, 2003,Vol.63, p.1483-1489.

Non-patent Reference 3: Suzuki Y. et al., “Molecular Cell”, 2001, Vol.8,p.613-621.

Non-patent Reference 4: Hegde R. et al., “The Journal of BiologicalChemistry”, 2002, Vol.277, p.432438.

Non-patent Reference 5: Martins L. M. et al., “The Journal of BiologicalChemistry”, 2002, Vol.277, p.439444.

Non-patent Reference 6: “Jikken Igaku (Experimental Medicine)”,2002,Vol.20, No.1, p.73-75.

Non-patent Reference7: Conti L. et al., “Nature Neuroscience”, 2001,Vol.4, p.579-586.

Non-patent Reference8: “Seikagaku (Biochemistry)”,2001, Vol.73, No.11,p.1322-1325.

Non-patent Reference9: Kaufinan R. J. et al., “The Journal of ClinicalInvestigation”, 2002, Vol.110, p.1389-1398.

Non-patent Reference 10: Haze K. et al., “The Biochemical Journal”,2001, Vol.355, p.19-28.

Non-patent Reference 11: Ulmer K. M., “Science”, 1983, Vol.219,p.666-671.

Non-patent Reference 12: “PEPUTIDO GOUSEI”, Maruzen Co., Ltd., 1975.

Non-patent Reference 13: “Peptide Synthesis”, Interscience, New York,1996.

DISCLOSURE OF THE INVENTION

[Problem To Be Solved By The Invention]

Today, caspase independent cell death in neurodegenerative diseases hasbeen reported in many papers. Meanwhile, it was reported that neuralcell death is caused through endoplasmic reticulum stress in brainischemia. HtrA2 has been considered to play an important role in themechanism for inducing caspase independent cell death. From these facts,it is believed that HtrA2 is likely to be a new target ofneurodegenerative disease and that inhibition of the neural cell deathdue to endoplasmic reticulum stress may become a novel target for drugdiscovery.

Under the circumstances where a mechanism of caspase independent celldeath caused by HtrA2 and a substrate of HtrA2 have not yet beenclarified, an object of the present invention is to find out a proteinthat interacts with HtrA2 and to provide a means that allows preventionand/or treatment of disorders attributable to the degradation of theprotein by HtrA2.

[Means For Solving The Problem]

The present inventors have concentrated intensively efforts to solve theaforementioned problem. We predicted in-silico that HtrA2 interacts withCREBL1 and SHC 1, and then found experimentally that CREBL1, ATF6 thatis a family member of CREBL 1, and SHC3 that is a family member of SHC1,were degraded by active HtrA2. Thus, we achieved the present invention.

Namely, the present invention relates to a method for inhibiting neuralcell death, comprising inhibiting the degration by HtrA2 (hightemperature requirement protein A2) of at least one of SHC3 (srchomology 2 domain containing transforming protein C3), ATF6 (activatingtranscription factor 6), and CREBL1 (cAMP responsive element bindingprotein-like 1).

The present invention also relates to a method for inhibiting neuralcell death, comprising using one or more compounds that inhibit thedegradation by HtrA2 of at least one of SHC3, ATF6 and CREBL1.

The present invention further relates to the aforementioned method forinhibiting neural cell death, in which the neural cell death is neuralcell death attributable to brain ischemia

The present invention still further relates to a method for preventing,treating or controlling brain ischemia or neurodegenerative disease,comprising inhibiting the degradation by HtrA2 of at least one of SHC3,ATF6 and CREBL1.

The present invention also relates to the aforementioned preventing,treating, or controlling method, in which the neurodegenerative diseaseis Alheimer's disease, Parkinson's disease, polyglutamine disease, priondisease, or amyotrophic lateral sclerosis.

The present invention further relates to a method of identifying acompound that inhibits the degradation by HtrA2 of at least one of SHC3,ATF6 and CREBL1, comprising contacting HtrA2 and at least one of SHC3,ATF6 and CREBL1 with a compound under conditions that allow thedegradation by HtrA2 of at least one of SHC3, ATF6 and CREBL1;introducing a system using a signal and a marker capable of detecting atleast one of SHC3, ATF6 and CREBL1; detecting the presence or absenceand/or change of the signal and the marker; and determining whether thecompound inhibits the degradation of at least one of SHC3, ATF6 andCREBL1.

The present invention still further relates to a method of identifying acompound that inhibits the degradation by HtrA2 of at least one of SHC3,ATF6 and CREBL1, comprising contacting HtrA2 and at least one of SHC3,ATF6 and CREBL1 with a compound under conditions that allow thedegradation by HtrA2 of at least one of SHC3, ATF6 and CREBL1; detectingthe presence or absence of at least one of SHC3, ATF6 and CREBL1, and/ormeasuring the change of the amount thereof; or detecting the presence orabsence of the degradation product of at least one of SHC3, ATF6 andCREBL1, and/or measuring the change of the amount thereof; anddetermining whether the compound inhibits the degradation of at leastone of SHC3, ATF6 and CREBL1.

The present invention also relates to an agent for inhibiting neuralcell death, comprising one or more compounds that inhibit thedegradation by HtrA2 of at least one of SHC3, ATF6 and CREBL1.

The present invention further relates to the aforementioned agent forinhibiting neural cell death, wherein the neural cell death is neuralcell death attributable to brain ischemia.

The present invention still further relates to an agent for preventing,treating or controlling brain ischemia or neurodegenerative disease,comprising one or more compounds that inhibit the degradation by HtrA2of at least one of SHC3, ATF6 and CREBL1.

The present invention also relates to the aforementioned agent forpreventing, treating or controlling neurodegenerative disease, whereinthe neurodegenerative disease is Alzheimer's disease, Parkinson'sdisease, polyglutamine disease, prion disease, or amyotrophic lateralsclerosis.

The present invention further relates to a reagent kit, comprising atleast one selected from the group consisting of HtrA2, a polynucleotideencoding HtrA2, and a vector containing the polynucleotide encodingHtrA2; and at least one selected from the group consisting of SHC3,ATF6, CREBL1, a polynucleotide encoding at least one of SHC3, ATF6 andCREBL1, and a vector containing the polynucleotide encoding at least oneof SHC3, ATF6 and CREBL 1.

[Advantage Of The Invention]

According to the present invention, to inhibit the degradation by HtrA2of at least one of CREBL1, ATF6, and SHC3 enables to inhibit thereduction or disappearance of the amount of at least one of theseproteins, and as result, enables to inhibit cell death (for example,neural cell death) and to prevent, treat, and control diseasesaccompanied with cell death (such as brain ischemia andneurodegenerative diseases).

SHC3 is expressed specifically in mature central nerve cells and isconsidered to play an important role in the differentiation and survivalof neural cells (Non-Patent References 7 and 8). ATF6 and CREBL1 areconsidered to perceive the endoplasmic reticulum stress to inducetranscription of chaperon genes and involve in the recovery from theendoplasmic reticulum stress in cells and survival of the cells(Non-Patent References 9 and 10). From these facts, it is believed thatthe degradation by HtrA2 of at least SHC3, ATF6, and CREBL1 causes thereduction or disappearance of the amount of these proteins, and therebyinduces the enhanced cell death due to stress and the like, such asneural cell death Therefore, to inhibit the degradation by HtrA2 of atleast SHC3, ATF6, and CREBL1 and thereby to inhibit the reduction ordisappearance of the amount of these proteins enable to inhibit celldeath (such as neural cell death), and enable to prevent, treat, orcontrol of diseases accompanied with cell death (for example, brainischemia or neurodegenerative diseases).

According to the present invention, a method of identifying a compoundthat inhibits the degradation by HtrA2 of at least one of SHC3, ATF6 andCREBL1 can be also carried out. The compound obtained by theidentification method can be used for inhibiting cell death (forexample, neural cell death), diabetes, and for preventing, treating, orcontrolling a disease accompanied with cell death (such as brainischemia and neurodegenerative diseases).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-A shows that CREBL1 was degraded in vitro by active HtrA2 (matureHtrA2). The band of CREBL1 was significantly reduced with the reactionof active HtrA2 with CREBL1 for overnight (ON). On the other hand, thedegradation of CREBL1 by inactive HtrA2 (mature HtrA2 (S306A)) was notobserved (Example 2).

FIG. 1-B shows that ATF6 was degraded in vitro by active HtrA2 (matureHtrA2). The band of ATF6 was significantly reduced with the reaction ofactive HtrA2 with ATF6 for 4 hours (4 h) or for overnight (O/N). On theother hand, the degradation of ATF6 by inactive HtrA2 (mature HtrA2(S306A)) was not observed (Example 2).

FIG. 1-C shows that SHC3 (p64) was degraded in vitro by active HtrA2(mature HtrA2). The band of SHC3 (p64) was significantly reduced withthe reaction of active HtrA2 with SHC3 (p64) for four hours (4 h) orovernight (O/N). On the other hand, the degradation of SHC3 (p64) byinactive HtrA2 (mature HtrA2 (S306A)) was not observed (Example 2).

FIG. 2-A shows that CREBL1 was degraded in a cell by active HtrA2 mutant(mature HtrA2 (ΔAVPS) that lacks N-terminal four amino acid residues ofthe active HtrA2 (mature HtrA2); or mature HtrA2 (GVPS) with thesubstitution of alanine at the N-terminal amino acid of the active HtrA2with glycine) (upper panel). In the analysis using cells in which themature HtrA2 (ΔAVPS) or the mature HtrA2 (GVPS) was co-expressed withCREBL1, the band of CREBL1 was significantly reduced (upper panel). Onthe other hand, in the analysis using cells in which inactive HtrA2mutant (the mature HtrA2 S306 (ΔAVPS) or the mature HtrA2 S306 (GVPS))was co-expressed with CREBL1, reduction of the band of CREBL1 was notobserved (upper panel). The expression of each HtrA2 mutant in the cellswas almost same among the cells (Power panel) (Example 3).

FIG. 2-B shows that ATF6 was degraded in a cell by active HtrA2 mutant(mature HtrA2 (ΔAVPS) that lacks N-terminal four amino acid residues ofthe active HtrA2 (mature HtrA2); or mature HtrA2 (GVPS) with thesubstitution of alanine at the N-terminal amino acid of the active HtrA2with glycine) (upper panel). In the analysis using cells in which themature HtrA2 (ΔAVPS) or the mature HtrA2 (GVPS) was co-expressed withATF6, the band of ATF6 was significantly reduced (upper panel). On theother hand, in the analysis using cells in which inactive HtrA2 mutant(the mature HtrA2 S306 (ΔAVPS) or the mature HtrA2 S306 (GVPS)) wasco-expressed with ATF6, reduction of the band of ATF6 was not observed(upper panel). The expression of each HtrA2 mutant in the cells wasalmost same among the cells (lower panel) (Example 3) FIG. 2-C showsthat SHC3 (p64) was degraded in a cell by active HtrA2 mutant (matureHtrA2 (ΔAVPS) that lacks N-terminal four amino acid residues of theactive HtrA2 (mature HtrA2); or mature HtrA2 (GVPS) with thesubstitution of alanine at the N-terminal amino acid of the active HtrA2with glycine) (upper panel). In the analysis using cells in which themature HtrA2 (ΔAVPS) or the mature HtrA2 (GVPS) was co-expressed withSHC3 (p64), the band of SHC3 (p64) was significantly reduced (upperpanel). On the other hand, in the analysis using cells in which inactiveHtrA2 mutant (the mature HtrA2 S306 (ΔAVPS) or the mature HtrA2 S306(GVPS)) was co-expressed with SHC3 (p64), reduction of the band of SHC3(p64) was not observed (upper panel). The expression of each HtrA2mutant in the cells was almost same among the cells (lower panel)(Example 3)

FIG. 2-C shows that SHC3 (p52) was degraded in a cell by active HtrA2mutant (mature HtrA2 (ΔAVPS) that lacks N-terminal four amino acidresidues of the active HtrA2 (mature HtrA2); or mature HtrA2 (GVPS) withthe substitution of alanine at the N-terminal amino acid of the activeHtrA2 with glycine) (upper panel). In the analysis using cells in whichthe mature HtrA2 (ΔAVPS) or the mature HtrA2 (GVPS) was co-expressedwith SHC3 (p52), the band of SHC3 (p52) was significantly reduced (upperpanel). On the other hand, in the analysis using cells in which inactiveHtrA2 mutant (the mature HtrA2 S306 (ΔAVPS) or the mature HtrA2 S306(GVPS)) was co-expressed with SHC3.(p52), reduction of the band of SHC3(p52) was not observed (upper panel). The expression of each HtrA2mutant in the cells was almost same among the cells (lower panel)(Example 3)

FIG. 3-A shows that the degradation of CREBL1 by active HtrA2 (matureHtrA2) was detected in the study using N-terminal tagged-CREBL1 withbiotinylated lysine residue within the protein and detecting the biotin.The band of biotinylated CREBL1 was significantly reduced with thereaction of active HtrA2 with CREBL1 for 4 hours (4h) or overnight(O/N), and the band around 50 kDa was detected which is considered to bea band indicating the degradation product of CREBL1. On the other hand,the degradation of CREBL1 by inactive HtrA2 (mature HtrA2 (S306A)) wasnot observed, and the band around 50 kDa as above was not detectedExample 4).

FIG. 3-B shows that the degradation of SHC3 (p64) by active HtrA2(mature HtrA2) was detected in the study using N-terminal tagged- SHC3p64) with biotinylated lysine residue within the protein and detectingthe biotin. The band of biotinylated SHC3 (p64) was significantlyreduced with the reaction of active HtrA2 with SHC3 (p64) for 4 hours(4h) or overnight (O/N), and the bands around 70 kDa, 40 kDa and 30 kDawere detected which are considered to be the bands indicating thedegradation products of SHC3 (p64). On the other hand, the degradationof SHC3 (p64) by inactive HtrA2 (mature HtrA2 (S306A)) was not observed,and the bands around 70 kDa, 40 kDa and 30 kDa as above were notdetected (Example 4).

FIG. 3-C shows that the degradation of SHC3 (p52) by active HtrA2(mature HtrA2) was detected in the study using N-terminal tagged-SHC3(p52) with biotinylated lysine residue within the protein and detectingthe biotin. The band of biotinylated SHC3 (p52) was significantlyreduced with the reaction of active HtrA2 with SHC3 (p52) overnight(O/N), but a band that is considered to be the bands indicating adegradation product of SHC3 (p52) was not detected. On the other hand,the degradation of SHC3 (p52) by inactive HtrA2 (mature HtrA2 (S306A))was not observed (Example 4).

FIG. 4-A shows that the degradation of CREBL1 by active HtrA2 (matureHtrA2) was detected in the study using N-terminal tagged-CREBL1 withbiotinylated lysine residue within the protein and detecting the Tagligated at the N-terminal. The N-terminal tagged-CREBL1 wassignificantly reduced with the reaction of active HtrA2 with CREBL1 for4 hours (4 h) or overnight (O/N). The band around 50 kDa that wasdetected in the study shown in FIG. 3, which is considered to be a bandindicating the degradation product of CREBL1, was not detected usinganti-Tag antibody. On the other hand, the degradation of CREBL1 byinactive HtrA2 (mature HtrA2 (S306A)) was not observed (Example 4).

FIG. 4-B shows that the degradation of SHC3 (p64) by active HtrA2(mature HtrA2) was detected in the study using C-terminal tagged-SHC3(p64) with biotinylated lysine residue within the protein by detectingthe Tag ligated at the C-terminal. The band of C-terminal tagged-SHC3(p64) was significantly reduced with the reaction of active HtrA2 withSHC3 (p64) for 4 hours (4 h) or overnight (O/N), and the bands around 70kDa, 40 kDa, and 30 kDa which are considered to be a band indicating thedegradation products of SHC3 p64), were detected. On the other hand, thedegradation of SHC3 p64) by inactive HtrA2 (mature HtrA2 (S306A)) wasnot observed (Example 4).

FIG. 4-C shows that the degradation of SHC3 (p52) by active HtrA2(mature HtrA2) was detected in the study using C-terminal tagged-SHC3(p52) with biotinylated lysine residue within the protein by detectingthe Tag ligated at the C-terminal. The band of C-terminal tagged-SHC3(p52) was significantly reduced with the reaction of active HtrA2 withSHC3 (p52) overnight (O/N), and the band around 40 kDa which isconsidered to be a band indicating the degradation product of SHC3(p52), was detected. On the other hand, the degradation of SHC3 (p52) byinactive HtrA2 (mature HtrA2 (S306A)) was not observed (Example 4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims the benefit of priority from JapanesePatent Application No. 2003-342588, which is incorporated herein byreference in its entirety.

In the present specification, the term “polypeptide” may be used as ageneric term which includes the followings: an isolated or a syntheticfull-length protein; an isolated or a synthetic full-length polypeptide;and an isolated or a synthetic full-length oligopeptide. A protein, apolypeptide or an oligopeptide used herein comprises two or more aminoacids that are bound to each other by peptide bond or modified peptidebond. Herein after, an amino acid may be represented by a single letteror by three letters.

In the present invention, the interaction of HtrA2 with CREBL1 and withSHC1 was predicted in-silico according to the method described in thepamphlet of Intermational Publication No. WO 01/67299. Further, it wasrevealed by way of experiment, for the first time, that HtrA2 degradesCREBL1, ATF6 belonging to the CREBL1 family and SHC3 belonging to theSHC1 family.

SHC3 is also referred to as N-Shc or ShcC and is specifically expressedin the mature central nerve cells. SHC3 is considered to transmit asignal from Trk family, a receptor tyrosine kinase, activated by theneurotrophic factors (such as NGF and EGF) and to play an important rolein the differentiation and survival of neural cells via PI3K-Akt-Badpathway. In addition, it has been also reported that cells in which onlySH domain of SHC3 (dominant negative) was expressed exhibited cell deathand showed suppressed neurite extension (Non-Patent References 7 and 8).

CREBL1 is one of ATF6 family and is also referred to as ATF6β. It hasbeen known that locates in endoplasmic reticulum and the portion thereoftranslocates into nucleus upon endoplasmic reticulum stress afterprocessing by S1P and S2P to work as a transcription factor.

It has been known that both CREBL1 and ATF6 perceive the endoplasmicreticulum stress to induce transcription of chaperon genes, and involvein the recovery of the stress in cells and the survival of the cells(Non-Patent References 9 and 10). In addition, there are some reportsdemonstrating the relation between endoplasmic reticulum stress andneurodegenerative diseases (Non-Patent Reference 1).

From these facts, it is believed that the degradation by HtrA2 of atleast one of SHC3, ATF6, and CREBL1 causes the reduction ordisappearance of the amount of these proteins, and thereby induces thedisruption of signal pathway for the development and survival of neuralcells, resulting in the enhanced neural cell death.

Accordingly, to inhibit the degradation by HtrA2 of at least one ofSHC3, ATF6, and CREBL1 enables to inhibit cell death. Further to inhibitthe degradation by HtrA2 of at least one of SHC3, ATF6, and CREBL1enables the prevention and/or treatment of diseases accompanied withcell death. For example, to inhibit the degradation by HtrA2 of at leastone of SHC3, ATF6, and CREBL1 enables to inhibit neural cell death, suchas the neural cell death due to endoplasmic reticulum stress.Furthermore, it can be conducted to prevent, treat, or control thediseases accompanied with cell death, for example, neurodegenerativediseases and brain ischemia In addition, it is believed that an agentfor inhibiting the degradation by HtrA2 of at least one of SHC3, ATF6,and CREBL1 may become a pharmaceutical agent for treatingneurodegenerative diseases and brain ischemia.

Based on these findings thus obtained, the present invention provides amethod for inhibiting the degradation by HtrA2 of at least one of SHC3,ATF6, and CREBL1. The present invention also provides a method forinhibiting neural cell death, comprising inhibiting the degradation byHtrA2 of at least one of SHC3, ATF6, and CREBL. The method forinhibiting neural cell death according to the present invention may beconducted preferably in vitro. Further, the present invention provides amethod for preventing, treating or controlling diseases attributable tothe degradation of at least one of SHC3, ATF6, and CREBL1.

A method for inhibiting the degradation of at least one of SHC3, ATF6,and CREBL1 can be carried out by inhibiting the function of HtrA2. Forexample, the method can be carried out by inhibiting the interaction ofHtrA2 with SHC3, ATF6, or CREBL1. Alternatively, the method can becarried out by inhibiting the enzyme activity of HtrA2. The inhibitionof the degradation of at least one of SHC3, ATF6, and CREBL1 can beachieved by inhibiting the function of HtrA2 preferably in an in vitrosample comprising HtrA2 and at least one of SHC3, ATF6, and CREBL1.Herein, such a substance that has an inhibitory effect (for example, apeptide, an antibody and a low molecular weight compound described laterthat have a competitive inhibitory effect) is referred to “aninhibitor”. “Interaction of HtrA2 with SHC3, ATF6, or CREBL1” asdescribed herein means that HtrA2 and SHC3, ATF6, or CREBL1 affect eachother in a certain manner, and as a specifically result, HtrA2 maydegrade SHC3, ATF6, or CREBL1. The term “in a certain manner” includes“by binding to”, “by contacting with”, or “by being adjacent to”, andmay be any manner as long as it allows their affection each other.

An inhibition of the degradation by HtrA2 of at least one of SHC3, ATF6,and CREBL1 can be carried out using a compound that inhibits theinteraction of at least one of SHC3, ATF6, and CREBL1 with HtrA2. Such acompound may be, for example, a polypeptide comprising the amino acidsequence of a site, in each of amino acid sequences of SHC3, ATF6,CREBL1 or HtrA2, where SHC3, ATF6, or CREBL1 interacts with HtrA2. Inparticular, such a polypeptide derived from SHC3, ATF6, or CREBL1 whichserves as the substrate of HtrA2 can competitively inhibit theinteraction of SHC3, ATT6, or CREBL1, with HtrA2, and can inhibit thedegradation by HtrA2 of these proteins. Such a polypeptide can beobtained by designing polypeptides based on the amino acid sequence ofHtrA2, SHC3, ATF6, or CREBL1, synthesizing them by peptide synthesismethods well-known in the art, and selecting a polypeptide that inhibitsthe degradation of at least one of SHC3, ATF6, or CREBL1 by HtrA2. Forexample, the polypeptide comprising the amino acid sequence of adegradation site by HtrA2 in each of amino acid sequences of SHC3, ATF6,or CREBL1 may be preferably used for such a compound. A polypeptidehaving an amino acid sequence derived from the thus specifiedpolypeptide, in which a mutation such as a deletion, substitution,addition or insertion of one to several amino acids was introduced, isalso included in the scope of the present invention. A polypeptide thatinhibits the degradation by HtrA2 of SHC3, ATF6, or CREBL1, ispreferable for such a polypeptide into which the mutation is introduced.A polypeptide having the mutation may be a naturally existingpolypeptide or a polypeptide in which a mutation was introduced.Techniques for introducing a mutation such as a deletion, substitution,addition or insertion are known. For example, the Ulmer technique(Non-patent Reference 11) may be utilize. When introducing a mutation asdescribed above, in view of avoiding a change in the fundamentalproperties (such as physical properties, function, physiologicalactivity, and immunological activity) of the polypeptide, mutualsubstitution among homologous amino acids (polar amino acids, non-polaramino acids, hydrophobic amino acids, hydrophilic amino acids,positive-charged amino acids, negative-charged amino acids and aromaticamino acids or the like) may be readily conceived. Furthermore, theseusable polypeptides can be modified to the extent that no significantfunctional change is involved, such as modification of its constituentamino group or carboxyl group and the like by an amidation and the like.

The aforementioned polypeptide can be produced by common methods thatare known in peptide chemistry. A method described in the References(Non-patent documents 12 and 13), for example, may be used, although themethods are not limited thereto, and known methods can be broadlyutilized. Particularly, a peptide synthesis method using an ordinaryliquid phase method and solid phase method, such as, for example, theFmoc method, can be used. Moreover, an amino acid synthesizer that iscommercially available can be used for producing the polypeptide.Alternatively, a gene engineering technology can be also used forproducing the polypeptide. For example, a gene encoding a polypeptide ofinterest can be used to prepare a recombinant expression vector that canexpress the gene in a host cell, followed by transfection of therecombinant expression vector into a suitable host cell such as E. colito obtain a transformant Culturing the transformant and collecting thepolypeptide of interest from the obtained culture product achieves theproduction of the polypeptide.

An inhibition of the degradation by HtrA2 of SHC3, ATF6, or CREBL1 canbe carried out using an antibody that recognizes HtrA2, SHC3, ATF6, orCREBL1 and inhibits the degradation by HtrA2 of SHC3, ATF6, or CREBL1.Such an antibody can be produced by known methods for preparing anantibody using HtrA2, SHC3, ATF6, or CREBL1, a polypeptide comprisingthe amino acid sequence of an interaction site of SHC3, ATF6, or CREBL1with HtrA2, or the like as an antigen.

An inhibition of the degradation by HtrA2 of SHC3, ATF6, or CREBL1 canbe carried out using a compound that inhibits the enzyme activity ofHtrA2, preferably using a compound that specifically inhibits the enzymeactivity of HtrA2. Such a compound can be obtained, for example, usingat least one of SHC3, ATF6, and CREBL1 as a substrate by identifying acompound that inhibits the degradation of the substrate. “Specificallyinhibit” denotes that “inhibit HtrA2 strongly, but does not inhibit orweakly inhibit other enzymes”.

A method of identifying a compound that inhibits the degradation byHtrA2 of at least one of SHC3, ATF6, and CREBL1 can be constructed byutilizing a known pharmaceutical screening system. For example, acompound that inhibits the degradation by HtrA2 of at least one of SHC3,ATF6, and CREBL1 can be identified by selecting conditions that allowfor the degradation by HtrA2 of at least one of SHC3, ATF6, and CREBL1,contacting a compound to be tested (hereinafter, referred to the testcompound) with HtrA2 and/or at least one of SHC3, ATF6, and CREBL1 underthe conditions, employing a system that uses a signal and/or a markercapable of detecting the degradation of at least one of SHC3, ATF6, andCREBL1, and then detecting the presence, the absence or the change ofthe signal and/or the marker. The condition for allowing the degradationby HtrA2 at least one of SHC3, ATF6, and CREBL1 may be a condition invitro or in vivo. For example, a cell in which HtrA2 is coexpressed withat least one of SHC3, ATF6, and CREBL1 may be used. The co-expression inthe cell can be accomplished by using an appropriate vector containing apolynucleotide encoding SHC3, ATF6, or CREBL1 and an appropriate vectorcontaining a polynucleotide encoding HtrA2, and transfecting them intothe cell by a conventional genetic engineering method. The vectorcontaining a polynucleotide encoding SHC3, ATF6, or CREBL1 can be usedsolely or in combination thereof to transfect the cell. The cells maybe, for example, a eukaryotic cell, preferably an animal cell, morepreferably a cultured animal cell line, further preferably a culturedmammal cell line. Contact of the test compound with HtrA2 and/or atleast one of SHC3, ATF6, and CREBL1 may be conducted prior to adegradation reaction of at least one of SHC3, ATF6, and CREBL1 by HtrA2,or may be conducted by allowing the test compound to coexist in thedegradation reaction. As used herein, the term “marker” refers to achemicals that can not be detected itself by physical properties orchemical propertied, but can generate the aforementioned signal via achemical reaction and can be detected indirectly by using the physicalproperties or chemical properties of the generated signal as anindicator. As a signal and/or marker, the followings maybe used: areporter gene (such as chloramphenicol acetyl transferase gene),radioisotope, tag peptides (such as His-tag, Myc-tag, HA-tag, FLAG-tagor Xpress-tag), enzymes such as GST, biotin and fluorescent protein. Asignal and/or marker are not limited to these specific examples, and anylabeling chemicals generally used in a method of identifying compoundsmay be utilized. Methods for detecting these signals or markers areknown to those skilled in the art. As a convenient method, thedegradation of at least one of SHC3, ATF6, and CREBL1 by HtrA2 can bedetected by determining the presence, the absence and/or the change ofthe amount of these proteins or the amount of degradation products ofthese proteins. Determination of the amount of these proteins or theamount of degradation products of these proteins can be carried outusing a known method for detecting a protein or peptide, such as, forexample, Western blotting.

All of HtrA2, SHC3, ATF6, and CREBL1 are known proteins and disclosed inGenBank with the accession numbers NM_(—)013247, NM_(—)06848,NM_(—)007348, and NM_(—)00438, respectively.

The amino acid sequence of HtrA2 used in the Examples is shown in SEQ IDNO: 4. The nucleotide sequence of HtrA2 DNA encoding the amino acidsequence set forth in SEQ ID NO: 4 is shown in SEQ ID NO: 3. Thepolypeptide shown by the amino acid sequence set forth in SEQ ID NO:4 isa mature HtrA2. The mature HtrA2 denotes a mature protein that isgenerated from HtrA2 precursor protein (SEQ ID NO: 2) by cleavage of itsN-terminal 133 amino acid residues and has a protease activity.Hereinafter, the HtrA2 with protease activity may be referred to as anactive HtrA2. Further, a protein (SEQ ID NO: 8, which may be referred toas mature HtrA2 (ΔAVPS)) that lacks the N-terminal four amino, acidresidues (AVPS) in the mature HtrA2, or a protein (SEQ ID NO: 10, insome cases referred to as mature HtrA2 (GVPS)) with the substitution ofalanine among the N-terminal four amino acid residues of the matureHtrA2 with glycine may be used as active HtrA2. Such HtrA2 with anintroduced mutation can be used as active HtrA2 since there is no changein the protease activity. In the meantime, HtrA2 without proteaseactivity may be in some cases referred to as inactive HtrA2. Theinactive HtrA2 may be, for example, HtrA2 mutant without proteaseactivity resulting from a mutation of the amino acid residue at the sitenecessary for protease activity of HtrA2 in the amino acid sequence ofHtrA2. The site necessary for protease activity of HtrA2 includes aprotease activity domain, more preferably the 174^(th) , serine residueof mature HtrA2 (SEQ ID NO: 4) (which is corresponding to the 306^(th)residue of the precursor protein (SEQ ID NO: 2)). More specifically, theinactive HtrA2 can be, for example, HtrA2 mutant (SEQ ID NO: 6, whichmay be referred to as mature HtrA2 (S306A)) with a substitution of the174^(th) serine residue of mature HtrA2 (which is corresponding to the306^(th) residue of the precursor protein (SEQ ID NO: 2)) with alanine.Further, a protein (SEQ ID NO: 12, in some cases referred to as matureHtrA2 (S306A, ΔAVPS)) that lacks the N-terminal four amino acid residues(AVPS) in the mature HtrA2 (S306A), or a protein (SEQ ID NO: 14, in somecases referred to as mature HtrA2 (S306A, GVPS)) with the substitutionof alanine among the N-terminal amino four amino acid residues of themature HtrA2 with glycine may be used as inactive HtrA2.

The amino acid sequence of SHC3 used in the Examples is shown in SEQ IDNO: 16. Hereinafter, SHC3 protein shown by the amino acid sequence setfor in SEQ ID NO: 16 may be sometimes referred to SHC3 p64). Thenucleotide sequence of SHC3 (p64) DNA is shown in SEQ ID NO: 15. It wasfound that the nucleotide sequence set for in SEQ ID NO: 15 had adifference in six nucleotides compared with the nucleotide sequence ofSHC3 disclosed in accession number NM_(—)01648 (see Example 2). Inaddition, SHC3 (p52) that is a splicing variant of SHC3 was used in thepresent invention. SHC3 (p52) is a protein that is translated from thesecond ATG (360 bases down stream from the first ATG) within the codingregion of SHC3 gene.

The amino acid sequence of ATF6 used in the Examples is shown in SEQ IDNO: 20. In addition, the nucleotide sequence of ATF6 DNA is shown in SEQID NO: 19. It was found that the nucleotide sequence set forth in SEQ IDNO: 19 had a difference in fifteen nucleotides compared with thenucleotide sequence of ATF6 disclosed in accession number NM_(—)007348(see Example 2).

The amino acid sequence of CREBL1 used in the Examples is shown in SEQID NO: 18. Furthermore, the nucleotide sequence of CREBL1 DNA is shownin SEQ ID NO: 17. It was found that the nucleotide sequence set forth inSEQ ID NO: 17 had a difference in one nucleotide compared with thenucleotide sequence of CREBL1 disclosed in accession number NM_(—)00438(see Example 2).

In the present invention, HtrA2, SHC3, ATF6 and CREBL1, as well as eachgene that encodes each of these proteins, are not limited to proteins orgenes that are shown by the above-exemplified amino acid sequences orthe above-exemplified nucleotide sequences, and includes generallyreported HtrA2, SHC3, ATF6 and CREBL1.

HtrA2, SHC3, ATF6 and CREBL1 used in the present invention can be thoseprepared from cells in which these are expressed by means of geneticengineering techniques, or from any biological samples, or can be theproducts of cell-free synthesis systems or the chemical synthesisproducts. These can be subsequently further purified for use. Further,cells in which one or more of HtrA2, SHC3, ATF6 and CREBL1 are expressedby means of genetic engineering techniques can be used. Moreover, HtrA2,SHC3, ATF6 and CREBL1 can be labeled by ligating a different type ofprotein or polypeptide thereto at the N-terminus or the C-terminus,directly or indirectly via a linker peptide and the like, by means of,for example, genetic engineering techniques, as long as it has noinfluence upon the interaction of HtrA2 with SHC3, ATF6 or CREBL1, andupon the function of these proteins, such as protease activity of HtrA2,the feature of SHC3, ATF6 and CREBL1 as an enzyme substrate. A differenttype of protein or polypeptide may be, for example, β-galactosidase, animmunoglobulin Fc fragment (such as IgG) and a tag peptide (such asHis-tag, Myc-tag, HA-tag, FLAG-tag, or Xpress-tag).

A polynucleotide encoding HtrA2, SHC3, ATF6 or CREBL1 can be preparedfrom a human cDNA library by known genetic engineering techniques. Avector containing the polynucleotide that encodes HtrA2, SHC3, ATF6 orCREBL1 can be obtained by introducing the polynucleotide using knowngenetic engineering techniques into a suitable expression vector, suchas a vector derived from a bacterial plasmid.

A test compound may be, for example, a compound derived from a chemicallibrary or natural products, as well as a compound obtained by drugdesign based on the polynucleotide structure or tertiary structure ofHtrA2, SHC3, ATF6 or CREBL1. Alternatively, a compound obtained by drugdesign based on the structure of a polypeptide that comprises an aminoacid sequence of an interacting site with HtrA2 or of a cleavage site byHtrA2 in the amino acid sequence of at least one selected from the groupconsisting of SHC3, ATF6 and CREBL1 is also suitable as a test compound.

The method of identifying a compound according to the present inventioncan be specifically carried out by adding a test compound to the invitro protease assay (Example 2) using, for example, HtrA2 and at leastone of SHC3, ATF6 and CREBL1. Mature HtrA2 of an active type can beprepared from E-coli transfected with an appropriate vector contaning amature HtrA2 DNA obtained from the human kidney cDNA library by wellknown genetic engineering method. CREBL1 can be prepared from a culturedanimal cell line (for example, HEK293T cell) transfected with anappropriate vector containing CREBL 1 DNA obtained from the human braincDNA library by well known genetic engineering method. SHC3 can beprepared from a cultured animal cell line (for example, HEK293T cell)transfected with an appropriate vector containing SHC3 DNA obtained fromthe human brain cDNA library by well known genetic engineering method.ATF6 can be prepared from an animal cell (for example, HEK293T cell)transfected with an appropriate vector containing ATF6 DNA obtained fromthe human mammary gland cDNA library by well known genetic engineeringmethod. HtrA2, SHC3, ATF6 and CREBL1 are contained in the solublefractions of lysates of the cells in which these proteins arerespectively expressed. SHC3, ATF6 and CREBL1 are preferably expressedas a protein ligated with a tag peptide at the N-terminal or C-terminal,and are favorably purified for use by means of immuno-precipitationusing an antibody against the tag peptide. For example, these proteinsare subjected to immuno-precipitation using an antibody against the tagpeptide, and then captured on a resin (such as protein G sepharose) foruse. The in vitro protease assay can be carried out by adding matureHtrA2 to SHC3, CREBL1, or ATF6 captured on a resin, reacting them eachother at 37° C. overnight, and detecting these proteins by Westernblotting. With regard to SHC3 and ATF6, the protease assay may beperformed with 4 hours reaction, since SHC3 and ATF6 are degraded bymature HtrA2 in the reaction at 37° C. for 4 hours. Detection by Westernblotting can be performed using an antibody against a tag peptideligated to SHC3, CREBL1, or ATF6. Since SHC3, CREBL1, or ATF6 isdegraded by HtrA2, the amount of these proteins detected by Westernblotting is reduced or disappeared compared with a case without addingHtrA2. In the case that the reduction or disappearance of the amount ofSHC3, CREBL1, or ATF6 is inhibited by adding a compound to the reactionof mature HtrA2 with SHC3, CREBL1, or ATF6 in comparison with a casewithout adding a compound, it can be judged that the test compoundinhibits the degradation by HtrA2 of SHC3, CREBL1, or ATF6. The testcompound may be previously contacted with SHC3, CREBL1, or ATF6, ormature HtrA2, and then the reaction of the mature HtrA2 with SHC3,CREBL1, or ATF6 may be conducted.

Further, the method of identifying a compound according to the presentinvention can be specifically carried out by adding a test compound tothe intracellular protease assay (Example 3) using, for example, such acell in which at least one of SHC3, CREBL1, and ATF6 are co-expressedwith HtrA2. Co-expression of at least one of SHC3, CREBL 1, and ATF6with HtrA2 can be performed by transfecting a cultured animal cell line(for example, HEK293T cell) with an appropriate vector containing eachof SHC3 DNA, CREBL1 DNA, and ATF6 DNA prepared as mentioned above and anappropriate vector contaning HtrA2 DNA by a well known geneticengineering method. SHC3, CREBL1, and ATF6 are preferably expressed asthe proteins having tag peptides ligated to the N-terminal or C-terminalof these proteins, in order to detect the proteins easily. Active HtrA2is used for HtrA2. The active HtrA2 suitably used are mature HtrA2,preferably mature HtrA2 (ΔAVPS) that lacks N-terminal four amino acidresidues (AVPS) of mature HtrA2, or mature HtrA2 (GVPS) that has asubstitution of alanine among the N-terminal four amino acid residueswith glycine. It has been reported that N-terminal four amino acidresidues (AVPS) of mature HtrA2, which is a binding motif to APs(Inhibitor of apoptosis proteins) family protein acting for inhibitingcell death, bind to IAPs to inhibit the action, thereby to acceleratecaspase dependent cell death. Since this action may be likely to give aninfluence upon the protease assay in a cell, it is preferable to usemature type HrtA2 (ΔAVPS) or mature HtrA2 (GVPS) which does not bind toIAPs. The intracellular protease assay can be performed by culturing acell in which at least one of SHC3, CREBL1, and ATF6 are co-expressedwith HtrA2 at 37° C. for 48 hours, together with a test compound, andthen lysing the cell to detect SHC3, CREBL1, or ATF6 containing in thecell lysate by Western blotting. Detection by Western blotting can becarried out using an antibody against the tag peptide ligated to SHC3,CREBL1, or ATF6. Since SHC3, CREBL1, or ATF6 is degraded by HtrA2, theamount of these proteins detected by Western blotting is reduced ordisappeared compared with a case without adding HtrA2. In the case thatthe reduction or disappearance of the amount of SHC3, CREBL1, or ATF6 isinhibited by adding a compound in comparison with a case without addinga compound, it can be judged that the test compound inhibits thedegradation by HtrA2 of SHC3, CREBL1, or ATF6.

The method of identifying a compound according to the present inventionis not limited to the specific examples such as the aforementioned invitro protease assay or the intracellular protease assay, and anidentification method well known for a pharmaceutical screening may beused.

A compound obtained by the method of identifying a compound according tothe present invention can be utilized as an agent for inhibiting thedegradation by HtrA2 of at least one of SHC3, CREBL1, and ATF6. Thecompound can be selected in consideration of a balance between thebiological usefulness and toxicity to prepare a pharmaceuticalcomposition. In preparation of the pharmaceutical composition, thecompound may be used alone or in combination.

The compound that inhibits the degradation by HtrA2 of at least one ofSHC3, CREBL1, and ATF6 can be used as an active ingredient for an agentfor inhibiting cell death due to the degradation by HtrA2 of at leastone of SHC3, CREBL1, and ATF6. Preferably, the compound is useful for anagent for inhibiting cell death due to endoplasmic reticulum stress.More preferably, the compound is useful for an agent for inhibitingneural cell death, still more preferable for an agent for inhibitingneural cell death due to endoplasmic reticulum stress. These inhibitorscan be used for conducting a method for inhibiting cell death due toendoplasmic reticulum stress. Preferably, the inhibitors can be used forconducting a method for inhibiting neural cell death due to endoplasmicreticulum stress or brain ischemia

The compound that inhibits the degradation by HtrA2 of at least one ofSHC3, CREBL1, and ATF6 can be used for preparing an agent forpreventing, controlling and/or treating diseases attributable to thedegradation by HtrA2 of at least one of SHC3, CREBL1, and ATF6. Such adisease can be, for example, a disease accompanied with neural celldeath, such as Alzheimer's disease, Parkinson's disease, polyglutaminedisease, prion disease, or amyotrophic lateral sclerosis (ALS). Thesediseases exhibit in most cases a formation of aggregates of abnormalproteins in neural cells, and the relation between neural cell death andendoplasmic reticulum stress in these diseases has been reported(Non-Patent Reference 1). The aforementioned compound may also used forconducting a method for preventing, controlling and/or treating such adisease.

The agent for preventing, treating and/or controlling the diseasesaccording to the present invention can be a pharmaceutical agentprepared by using at least any one of the members consisting of theaforementioned compounds, degradation inhibitor, and cell deathinhibitor so as to contain the effective amount thereof. Generally, thepharmaceutical agent is preferably prepared as a pharmaceuticalcomposition that includes one or more kinds of a pharmaceutical carrier.

An amount of the effective ingredient containing in the pharmaceuticalpreparation can be suitably selected from wide range. In general, asuitable amount may fall within a range of approximately 0.00001 to 70wt %, preferably approximately 0.0001 to 5 wt %.

As a pharmaceutical carrier, the followings may be used: a filler,extender, binder, wetting agent, disintegrator, lubricant, diluent andexcipient that are generally used in accordance with the form of use ofthe formulation. These can be suitably selected and used in accordancewith the administration form of the preparation to be obtained.

More concretely, the pharmaceutical carrier may be water, apharmaceutically acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate,soluble dextan, sodium carboxymethyl starch, pectin, xanthan gum, acaciagun, casein, gelatin, agar, glycerin, propylene glycol, polyethyleneglycol, vaseline, paraffin, stearyl alcohol, stearic acid, human serumalbumin, mannitol, sorbitol and lactose. One or a combination of two ormore kinds of these may be suitably selected and used in accordance withthe dosage form of the pharmaceutical composition.

As desired, various components that may be used in conventional proteinpreparation, such as a stabilizer, bacteriocide, buffer agent,isotonizing agent, chelating agent, surfactant, or pH adjuster, may besuitably contained in the pharmaceutical composition.

As a stabilizer, the followings may be used: human serum albumin, commonL-amino acids, sugars and cellulose derivatives. These can be usedindependently or in combination with a surfactant and the like.Especially, use of these in such a combination may give increasedstability of an effective ingredient. An L-amino acid is notparticularly limited, and may be any one of glycine, cysteine, glutamicacid and the like. A sugar is not particularly limited, and may be anyone of monosaccharides (such as glucose, mannose, galactose andfructose), sugar alcohols (such as mannitol, inositol and xylitol),disaccharides (such as sucrose, maltose and lactose), polysaccharides(dextran, hydroxypropylstarch, chondroitin sulfate and hyaluronic acid),derivatives thereof and so on. A cellulose derivative is notparticularly limited, and may be any one of methylcellulose,ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethlcellulose, sodium carboxymethylcellulose and the like.

A surfactant is not particularly limited, and can be both an ionicsurfactant and a non-ionic surfactant As a surfactant, the followingsmay be used: polyoxyethyleneglycol sorbitan alkyl ester system,polyoxyethylene alkyl ether system, sorbitan monoacyl ester system andfatty acid glyceride system.

As a buffer agent, the followings may be used: boric acid, phosphoricacid, acetic acid citric acid, ε-aminocaproic acid, glutamic acid and/ora salt thereof (for example, an alkaline metal salt and an alkali earthmetal salt, such as sodium salt, kalium salt, calcium salt and magnesiumsalt).

As an isotonizing agent, the followings may be used: sodium chloride,kalium chloride, sugars and glycerin.

As a chelating agent, sodium edentate and citric acid may be used.

The pharmaceutical agent and pharmaceutical composition according to thepresent invention can be used as a solution preparation. Alternatively,they can be freeze-died so as to be in good state in preservation andcan be used by dissolving them in water, a buffered solution containingsaline and the like, and so on and then adjusting to suitableconcentration at the time of using.

Suitable dosage ranges of the pharmaceutical composition are notparticularly limited, and can be determined according to the following:effectiveness of the ingredients contained therein; the administrationform; the route of administration; the type of disease; thecharacteristics of the subject (e.g., body weight, age, symptomaticconditions and whether being taking other pharmaceutical agent) and thejudgment of the physician in charge. In general, a suitable dosage mayfall, for example, within a range of about 0.01 μg to 100 mg per 1 kg ofthe body weight of the subject, and preferably within a range of about0.1 μg to 1 mg per 1 kg. However, a dosage may be altered usingconventional experiments for optimization of a dosage that are wellknown in the art. The aforementioned dosage can be divided foradministration once to several times a day. Alternatively, periodicadministration once every few days or few weeks can be employed.

When administering the pharmaceutical composition of the presentinvention, the pharmaceutical composition may be used alone or may beused together with other compounds or pharmaceutical agents necessaryfor the treatment.

In terms of a route of administration, it may be either systemicadministration or local administration. The route of administration thatis appropriate for a particular disease, symptomatic conditions, orother factors should be selected. For example, parenteral administrationincluding normal intravenous injection, intraarterial administration,subcutaneous administration, intracutaneous administration andintramuscular administration can be employed. Oral administration can bealso employed. Further, transmucosal administration or dermaladministration can be employed.

In terms of an administration form, various forms can be selected inaccordance with a treatment purpose. Typically, for example, anadministration form of solid formulation may be used, such as a tablet,a pill, powder, powdered drug, fine granule, granule or a capsule, aswell as an administration form of liquid formulation such as an aqueousformulation, ethanol formulation, suspension, fat emulsion, liposomeformulation, clathrate such as cyclodextrin, syrup and an elixir. Thesecan be further classified according to the administration route intooral formulation, parenteral formulation (drip injection formulation orinjection formulation), nasal formulation, inhalant formulation,transvaginal formulation, suppositorial formulation, sublingual agents,eye drop formulation, ear drop formulation, ointment formulation, creamformulation, transdermal absorption formulation, transmucosal absorptionformulation and the like, which can be respectively blended, formed andprepared according to conventional methods.

The present invention further provides a reagent kit including at leastone member selected from the group consisting of HtrA2, a polynucleotideencoding HtrA2, and a vector containing a polynucleotide encoding HtrA2;and at least one member selected from the group consisting of SHC3,ATF6, CREBL1, a polynucleotide encoding SHC3, ATF6, or CREBL1, and avector containing the polynucleotide encoding SHC3, ATF6, or CREBL1. Thereagent kit can be used, for example, in the identification method ofthe present invention.

The aforementioned reagent kit may include chemicals that are necessaryfor carrying out a determination, such as. a signal and/or a marker fordetecting the degradation of SHC3, ATF6 or CREBL1 by HtrA2, buffersolutions and salts. The reagent kit may also include chemicalss such asstabilizers and/or antiseptic agents. At the time of preparation,methods for preparation may be introduced in accordance with therespective chemicals to be used.

Hereinafter, the present invention may be explained more particularlywith examples; however, the present invention is not limited to thefollowing examples.

EXAMPLE 1

(In-silico search for proteins having a function to interact with HtrA2)

The prediction of proteins that have function to interact with HtrA2 wasconducted according to the method described in the pamphlet ofInternational Publication No. WO 01/67299. Concretely, the amino acidsequence of HtrA2 was decomposed into oligopeptides having apre-determined length in order to search in a database for proteinshaving the amino acid sequence of each of the oligopeptides, or havinghomologous amino acid sequences to these amino acid sequences. Then,local alignment was conducted between the proteins obtained and HtrA2 toidentify proteins having a high local alignment score that might becapable of interacting with HtrA2.

As a result of analysis, CREBL1 and SHC1 was identified as a proteinbeing predicted to have a function to interact with HtrA2.

EXAMPLE 2

(In vitro Protease Assay)

In order to demonstrate experimentally the interaction of HtrA2 withCREBL1, ATF6 that is a family CREBL1, or SHC3 that is a family SHC1, invitro protease assay was performed.

<Materials and Their Preparation>

In this Example, mature HtrA2 (SEQ ID NO: 4) and mature HtrA2 (S306A)(SEQ ID NO: 6) to each of which histidine (His)-tag was ligated at theC-terminal were used as active HtrA2 and inactive HtrA2, respectively.Further, SHC3 (p64) (SEQ ID NO: 16) and SHC3 (p52) to each of whichc-Myc-tag was ligated at the C-terminal, and CREBL1 (SEQ ID NO: 18) andATF6 (SEQ ID NO: 20) to each of which c-Myc-tag was ligated at theN-terminal were used as a protein that was predicted to interact withHtrA2.

1. Cloning of mature HtrA2 and mature HtrA2 (S306A), and preparation ofexpression plasmid

In order to obtain a gene encoding mature HtrA2 (a portion of precursorHtrA2 (DNA nucleotide sequence thereof and amino acid sequence encodedby the DNA are shown in SEQ ID NO: 1 and SEQ ID NO: 2) from 134^(th) to458^(th) amino acid residues), mature HtrA2 gene was amplified bypolymerase chain reaction (PCR) and cloned into pCR-BluntII-TOPO vector(Invitrogen). The PCR was carried out by using Human Kidney QUICK-ClonecDNA (Clontech) as atemplate, HtrA2-F1 primer (with NdeI site and ATG atthe 5′ side, SEQ ID NO: 21), HtrA2-RS primer (with hoI site instead oftermination codon, SEQ ID NO: 22), and KOD-plus (Toyobo) as DNApolymerase. The nucleotide sequence was determined by sequencer (AppliedBiosystems/Hitachi: ABI3100). The nucleotide sequence of mature HtrA2DNA obtained in this example, and amino acid sequence encoded by the DNAare shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. E-coliexpression plasmid for mature HtrA2 was obtained by digesting the clonedmature HtrA2 gene with NdeI and XhoI, and then integrating it intopET24b vector (Novagen).

It has been reported that a mutant (mature HtrA2 (S306A)) with asubstitution of 174^(th) serine of mature HtrA2 (which is correspondingto 306^(th) in precursor protein (SEQ ID NO: 2)) with alanine does notexhibit protease activity. Then, E-coli expression plasmid for matureHtrA2 (S306A) that is of inactive type was prepared using mature HtrA2expression plasmid as a template, with QuickChange XL Site-DirectedMutagenesis kit (Stratagene) using HtrA2-MF primer (SEQ ID NO: 23) andHtrA2-MR primer (SEQ ID NO: 24). The nucleotide sequence was determinedby sequencer. The nucleotide sequence of mature HtrA2 (S306A) DNAobtained in this example and amino acid sequence encoded by the DNA areshown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

2. Expression and puinfication of mature HtrA2 and mature HtrA2 (S306A)E-coli expression plasmid for HtrA2 was transfected into into E-coliBL21 Star (DE3) competent cell anvitrogen) to obtain a trasformant Thetransformant was incubated in LB culture medium (100 ml) at 26° C., andexpression induction of mature HtrA2 protein was carried out by addingisopropyl-1-thio-P-D-galactopyrano (IPTG) to the final concentration of0.1 mM when absorbance (OD) reached in arange of 0.68 to 0.81. Afterfurther incubation for overnight, E-coli that expressed mature HrLA2 wasseparated by centrifugation and collected. E-coli thus obtained wassuspended in lysis buffer A (Lysis buffer: phosphate buffered saline(PBS)/1% TritonX-100, 1 μg/ml pepstatin, 5 μM E64) followed by treatmentwith sonicator (15 sec×10 times) under ice-cooling to disrupt the fungusbody. The disrupted fungus body solution was subjected to centrifigation(15,000 rpm, 30 min, 4° C.) to separate into a soluble fraction(supernatant) and an insoluble fraction. Soluble fraction contaningmature HtrA2 was added to ProBondresin (Invitrogen: 1 ml prepacked) thathad been preequilibrated with 1% Triton X-100 in phosphate bufferedsaline (PBS), and mixed at 4° C. for 30 min, and then washed three timeswith washing buffer (20 mM sodium phosphate (pH 6.0), 500 mM NaCl).Mature HtrA2 adsorbed by the resin was eluted in stepwise by elutionbuffer (20 mM, sodium phosphate (pH 6.0) 500 mM NaCl) each containing50, 100, 200, 350, 500 mM imidazole. Each eluate was subjected toSDS-PASGE to identify the fraction containing mature HtrA2. As a result,mature HtrA2 was eluted by 350-500 mM immidazole. The fractioncontaining mature HtrA2 was dialyzed against 150 mM NaCl, 50 mM Tris-HCl(pH 7.5), followed by centrifugation to remove impurities. The resultantsupernatant was concentrated followed by subjecting to the quantitativemeasurement of protein by Coomassie Plus Protein Assay (PERCE) whilebovine serum albumin (BSA) was used as the standard protein. Similarly,expression and purification of mature HtrA2 (S306A) were performed.

3. Cloning of SHC3 (p64), SHC3 (p52), ATF6 and CREBL1, and preparationof expression plasmid SHC3 (p64) gene was amplified by PCR and clonedinto pCR4Blunt-TOPO vector invitrogen). The PCR was carried out usingHuman Brain QUICK-Clone cDNA (Clontech) as a template, SHC3F1 primer(with BamHI site and GCC immediately before ATG, SEQ ID NO: 25), SHC3R1primer (with XhoI site instead of the termination codon, SEQ ID NO: 26),and KOD-plus as DNA polymerase. The nucleotide sequence was determinedby the sequencer. It was found that the nucleotide sequence of SHC3(p64) DNA thus obtained in this example had a difference in sixnucleotides compared with the nucleotide sequence that had already beenregistered in GenBank with accession number NM_(—)016848. It wasrevealed that four nucleotides out of the six different nucleotidescoincided with the genome sequence: T173A (with amino acid change fromVal to Asp); G789A (without amino acid change); A811G (with amino acidchange from Thrto Ala); and A1661G (with amino acid change from Gln toArg). The other two different nucleotides were G291 C and A1338G withoutamino acid change. The nucleotide sequence of SHC3 DNA obtained in thisexample and the amino acid sequence encoded by the DNA are shown in SEQID NO: 15 and SEQ ID NO: 16, respectively. SHC3 animal cell expressionplasmid was prepared by digesting the cloned SHC gene with BamHI andXhoI followed by recombination into pCMV-Tag5.

SHC3 (p52), which is a splicing valiant of SHC3, is a protein that istranslated from the second ATG (360 bases down stream from the firstATG) within the coding region of SHC3 gene. The expression plasmid ofSHC3 (p52) was constructed by digesting the SHC3 (p64) plasmid that hadbeen cloned into pCMV-Tag5A with SacI and XhoI, followed by integratinga SacII-XhoI fragment into pCMV-Tag5A vector. The integrated SHC3 (p52)gene in the expression plasmid was confirmed by digestion withrestriction enzyme.

CREBL1 gene was amplified by PCR and cloned into pCMV-Tag 5. The PCR wascarried out using Human Brain cDNA as a template, 83-F primer (withEcoRI site and GCC immediately before ATG, SEQ ID NO: 27), 83-R primer(with XhoI site instead of the termination codon, SEQ ID NO: 28), andKOD-plus as DNA polymerase.

Then, CREBL1 gene was amplified by PCR and cloned into pCR-BluntII-TOPOvector. The PCR was carried out using the CREBL1 gene cloned intopCMV-Tag 5 as a template, ATF6-NF1 primer (with Bamfi site instead ofATG, SEQ ID NO: 29), ATF6-NR1 primer (with XhoI site following totermination codon, SEQ ID NO: 30), and KOD-plus as DNA polymerase. Thenucleotide sequence was determined by the sequencer. It was found thatthe nucleotide sequence obtained in this example had a difference in onenucleotide (T450C) compared with the nucleotide sequence of CREBL1 thathad already been registered in GenBank with accession numberNM_(—)00438. There is no change in the amino acid resulting from thisdifference. In the meantime, the termination codon was changed from TGAto TAA. It was confirmed that these differences were not due to PCRerrors. The nucleotide sequence of CREBL1 DNA obtained in this exampleand the amino acid sequence encoded by the DNA are shown in SEQ ID NO:17 and SEQ ID NO: 18, respectively. Animal cell expression plasmid forCREBL1 was prepared by digesting the cloned CREBL1 gene with BamHI andXhoI, and then integrated it into pCMV-Tag 3.

ATF6 gene was amplified by PCR using Mammary Gland QUICK-Clone cDNA(Clontech) as a template, ATF6Fn primer (SEQ ID NO: 31), ATF6Rn primer(SEQ ID NO: 32), and KOD-plus as DNA polymerase. Then, ATF6 gene wasfurther amplified by PCR using the ATF6 gene thus amplified, asatemplate, using ATF6F1L primer (with EcoRV site immediately before ATG,SEQ ID NO: 33), ATF6R1L primer (with XhoI site immediately after thetermination codon, SEQ ID NO: 34), and KOD-plus as DNA polymerase, andthen cloned into pCR4Blunt-TOPO vector Introgen). The nucleotidesequence was determined by the sequencer (SEQ ID NO: 19). It was foundthat the nucleotide sequence of ATF6 DNA thus obtained in this examplehad a difference in fifteen nucleotides compared with the nucleotidesequence that had already been registered in GenBank with accessionnumber NM_(—)007348. It was revealed that all of the fifteen differentnucleotides coincided with the genome sequence: T105C (without change inamino acid); T199A (accompanied with change in amino acid from Leu toMet); A201G (accompanied with change in amino acid from Leu to Met);C270T (without change in amino acid); A309G (without change in aminoacid); C433G (accompanied with change in amino acid from Pro to Ala);T469C (accompanied with change in amino acid from Ser to Pro); G1228A(accompanied with change in amino acid from Gly to Ser); A1230C(accompanied with change in amino acid from Gly to Ser); C1389T (withoutchange in amino acid); T1416C (without change in amino acid); T1491Awithout change in amino acid); T1538C (accompanied with change in aminoacid from Val to Ala); G1540C (accompanied with change in amino acidfrom Val to Leu); and 1896A (without change in amino acid). In themeantime, there is a description in SWISS-PROT about changes of allthese amino acids in terms of conflict The nucleotide sequence of ATF6DNA obtained in this example and the amino acid sequence encoded by theDNA are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively.

In order to integrate ATF6 gene into animal cell expression vector, ATF6gene was amplified by PCR using the aforementioned ATF6 gene having beencloned into pCR4Blunt-TOPO vector as a template, using ATF6F2L primer(with BgHI site immediately before ATG, SEQ ID NO: 35), ATF6R1L primer(SEQ ID NO: 34), and KOD-plus as DNA polymerase, and then cloned intopCR4Blunt-TOPO vector. The nucleotide sequence was determined bysequencer (SEQ ID NO: 19). ATF6 gene thus cloned was digested by BglIIand XhoI to integrate into animal cell expression vector, pCMV-Tag3,digested with BamHI and XhoI.

4. Expression of SHC3 (p64), SHC3 (p52), ATF6 and CREBL1 HEK293T cellswere cultured to 1.5×10⁶ cells/10 cm Dish. Next day, the expressionplasmid of SHC3 (p64), SHC3 (p52), CREBL1 or ATF6 (10 pg/Dish) wastransfected into HEK293T cells using FuGene 6 (Roche). Forty eight hourslater, cells were washed with PBS, followed by adding 1 ml of lysisbuffer B (50 mM Tris-HCl (pH 7.6),150 mM NaCl, 1% Triton X-100, 1%Nonidet P-40 (NP-40), Complete Mini-EDTA (ethylenediamine tetra-acetate)free), and left to stand on ice for 10 min. Then, cells were collectedwith a scraper, put into a 1.5 ml tube, subjected to sonication underice-oling (15 sec×6 times), left to stand on ice for 20 min, suspendedby pipetting, followed by subjecting centrifugation (15,000 rpm, 30 min,4° C.) to separate into a soluble fraction and an insoluble fraction.The soluble fractions were used as a test sample.

5. Determination of the activity of mature HtrA2 and mature HtrA2(S306A) Determination of the activity of mature HtrA2 and mature HtrA2(S306A) was carried out with casein zymography and with a protease assaywith casein sodium. Casein zymography was carried out according to theprotocol (Invitrogen). More concretely, mature HtrA2 and mature HtrA2(S306A) were mixed under non-reductive condition with equivalent volumeof 2×SDS sample buffer, left to stand at room temperature for 10 min,followed by separation with 4-16% zymogram (Blue casein) gel (ZymogramGel, Invitrogen). After completion of electrophoresis, they wererenatured with 2.5% Triton X-100, and then subjected to a degradationreaction of casein in the developing buffer at 37° C. for overnight

Meanwhile, aprotease assay using casein sodium as an substrate wascarried out in such that mature HtrA2 or mature HtrA2 (S306A) was mixedwith casein sodium in a reaction buffer (150 mM NaCl, 50 mM Tris-HCl (pH7.5)) so as to the concentration of the former might become 200 g/ml andthe concentration of the latter might become 400 μg/ml., and incubatedat 37° C. for reactions. A portion of the reaction solution was sampledat three hours and overnight after initiation of the reaction, mixedwith equivalent volume of 2×SDS sample buffer followed by boiling, andthen subjected to SDS-PAGE. After that, the degradation of casein sodiumwas detected by Coomassie Brilliant Blue (CBB) staining.

As a result of the aforementioned investigation, the degradation ofcasein by mature HtrA2 was observed, but the degradation of casein wasnot observed with mature HtrA2 (S306A). From these findings, it has beenconfirmed that mature HtrA2 is of active type having protease activity,but mature HtrA2 (S306A) is of inactive type not exhibiting proteaseactivity.

<Method>

SHC3 (p64), SHC3 (p52), ATF6 and CREBL1 each of which was captured onthe resin by immunoprecipitation was used in the experiments in order toeliminate the influence of proteinaceous impurities contained in thesoluble fraction. More specifically, 300 μl of the soluble fraction ofSHC3 (p64), SHC3 (p52), ATF6 or CREBL1 was dispensed to six tubes (Nos.1 to 6), followed by adding 20 μl of protein G sepharose 4 FF (Amersham)of 50% slurry that had been subjected to blocking with BSA, to each oftubes, subjected to overturned mixing at 4° C. for 1 hour, and thensubjected to centfigation (10,000 rpm, 10 sec, 4° C.) to collect thesupernatant, and thus the pre-treatment (pre-clean) was performed.

The resultant supernatant was mixed with 1.7 μl (2 μg) of anti-Mycantibody (Invitrogen) by overturned mixing at 4° C. for 3 hours,followed by adding 20 μl of protein G sepharose 4 FF that had beensubjected to blocking with BSA, and then subjected to overturned mixingat 4° C overnight. Thus, SHC3 (p64), SHC3 (p52), ATF6 and CREBL1 each ofwhich was captured on the protein G sepharose 4 FF were prepared.Subsequently, protein G sepharose 4 FF on which SHC3 (p64), SHC3 (p52),ATF6 or CREBL1 was captured was washed with 500 μl of washing buffer (50mM Tris-HCl (pH 7.5),150 mM NaCl, 0.01% Triton X-100) five times, and100 pi of 50 mM Tris-HCl (pH 7.5), 150 mMNaCl, 0.01% TritonX-100 wasadded to tubes Nos. 1 and 2, 100 μl of mature HtrA2 solution (50 μg/ml)was added to tubes Nos. 3 and 4, and 100 μl of mature HtrA2 (S306A)solution (50 μg/ml) was added to tubes Nos. 5 and 6, respectively. TubesNos. 1,3 and 5 were subjected to a reaction at 37° C. for four hours,tubes Nos.2,4,6 were subjected to a reaction overnight. After thereaction, the centrfigation was carried out to remove the supernatant.The precipitate was subjected to washing/centdigation for three timeswith 500 μl of washing buffer, followed by adding 80 μl of 2×SDS samplebuffer (containing 0.1% P-mercaptoethanol) and boiling. Ten μl of thesample thus obtained (80 μl) was subjected to SDS-PAGE and transfered toPVDF membrane onto detect SHC3 (64), SHC3 (p52), ATF6 and CREBL1 byWestern blotting. Anti-Myc antibody (9E10) (Sigma) was used as a primaryantibody for detection, and horseradish peroxidase (HRP) labeledanti-mouse IgG antibody (Cell Signaling) was used as a secondaryantibody. Detection was carried out using ECL Western Blotting DetectionSystem (Arnersham).

<Results>

It was observed that all of CREBL1, ATF6, and SHC3 (p64) were degradedby mature HtrA2 in vitro. As shown in FIG. 1-A, the band of CREBL1 wassignificantly reduced after the reaction of active HtrA2 with CREBL1 forovernight (O/N). As shown in FIG. 1-B, the band of ATF6 wassignificantly reduced after the reaction of active HtrA2 with ATF6 forfour hours (4 h) or overnight (O/N). As shown in FIG. 1-C, the band ofSHC3 (p64) was significantly reduced after the reaction of active HtrA2with SHC3 (p64) for four hours (4 h) or overnight (O/N). Further it wasobserved that SHC3 (p52) was also degraded by mature HtrA2.

Onthe other hand, the degradation of CREBL1, ATF6, and SHC3 (p64) byinactive HtrA2 (mature HtrA2 (S306A)) was not observed (FIG. 1-A, FIG.1-B and FIG. 1-C). Further, the degradation of SHC3 (p52) by inactiveHtrA2 (mature HtrA2 (S306A)) was not observed as well.

EXAMPLE 3

(Intracellular Protease Assay)

Interaction of HtrA2 with CREBL1, ATF6, SHC3 (p64) or SHC3 (p52) wasinvestigated by intracellular protease assay.

<Materials and Their Preparations>

It has been reported that N-terminal four amino acid residues (AVPS) ofmature HtrA2, which is a binding motif to IAPs (nhibitor of apoptosisproteins) family protein acting for inhibiting cell death, bind to IAPsto inhibit the action, thereby to accelerate caspase dependent celldeath. Since this action may be likely to give an influence upon theprotease assay in a cell. Therefore, in order to inhibit the interactionof mature HtrA2 or mature HtrA2 (S306A) with LAPs, those (ΔAVPS) thatlack the N-terminal four amino acid residues (AVPS) and those(AVPS→GVPS) with a substation of alanine among the four amino acidresidues with glycine were prepared for each of mature HtrA2 and matureHtrA2 (S306A). Each of these various types of HtrA2 was prepared asC-terminal FLAG-tagged protein for use.

1. Preparation of various types of HtrA2

In order to prepare mature HtrA2 mutants, each gene of mature HtrA2(ΔAVPS) and mature HtrA2 (GVPS) was amplified by PCR and cloned intopCR-BluntII-TOPO vector. The PCR was carried out using mature HtrA2 as atemplate, F1 and F2 primers (with SacI site immediately before ATG, SEQID NO: 36 and SEQ ID NO: 37, respectively) as the sense primer, HtrA2-RSprimer (SEQ ID NO: 22) as the anti-sense primer, and Pfu turbo(STRATAGENE) as the DNA polymerase. The nucleotide sequence wasdetermined by sequencer. Each of animal cell expression plasmids formature HtrA2 (ΔAVPS) and mature HtrA2 (GVPS) was prepared by digestingeach of the cloned mature HtrA2 (ΔAVPS) gene and the cloned mature HtrA2(GVPS) gene by SacI and XhoI, and then integrated it into pCMV-Tag4. Thenucleotide sequence of mature HtrA2 (ΔAVPS) DNA obtained in this exampleand the amino acid sequence encoded by the DNA are shown in SEQ ID NO: 7and SEQ ID NO: 8, respectively. Besides, the nucleotide sequence ofmature HtrA2 (GVPS) DNA and the amino acid sequence encoded by the DNAare shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

In order to prepare mature HtrA2 (S306A) mutants, with a same manner asmentioned above, each gene of mature HtrA2 (S306A, ΔAVPS) and matureHtrA2 (S306A, GVPS) was amplified by PCR using mature HtrA2 (S306A) as atemplate, and then cloned into pCR-BluntII-TOPO vector. Each of animalcell expression plasmids of mature HtrA2 (S306A, ΔAVPS) and mature HtrA2(S306A, GVPS) was prepared by digesting each of the cloned mature HtrA2(S306A, ΔAVPS) and the cloned mature HtrA2 (S306A, GVPS) by SacI andXhoI, and then integrated it into pCMV-Tag4. The nucleotide sequence ofmature HtrA2 (S306A, ΔAVPS) DNA obtained in this example and the aminoacid sequence encoded by the DNA are shown in SEQ ID NO: 11 and SEQ IDNO: 12, respectively. Besides, the nucleotide sequence of mature HtrA2(S306A, GVPS) DNA and the amino acid sequence encoded by the DNA areshown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

2. Determination of enzyme activity of various types of HtrA2

The enzyme activity of the various types of HtrA2 each of which wasexpressed in a cultured cell was determined by the protease assay usingcasein sodium as a substrate (see Example 2). As a result, it has beenrevealed that both mature HtrA2 (ΔAVPS) and mature HtrA2 (GVPS) are ofactive type having protease activity and that both mature HtrA2 (S306A,ΔAVPS) and mature HtrA2 (S306A, GVPS) are of inactive type withoutexhibiting protease activity.

3. Preparation of animal cell expression vectors for SHC3 (p64), SHC3(p52), ATF6 and CREBL1

The vectors that were prepared by the same procedures used in Example 2were used.

<Method>

HBEK293T cells were cultured to 1.5×10⁵ cells/6 cm Dish. Next day, theexpression plasmid of each protein in interest (SHC3 (p64), SHC (p52),ATF6, and CREBL1) was transfected into HEK293T cells together with theexpression plasmid of various types of HtrA2 using FuGene 6 (Roche).Each of the expression plasmid of various HtrA2 at 1 μg/Dish was addedin combination with any one of the expression plasmid of the protein ininterest at 1 μg/Dish. Forty eight hours later, the cells in which thevarious types of HtrA2 were co-expressed with SHC3 (p64) or SHC (p52)were added with 400 μl of lysis buffer B, and subjected tocentrifugation to collect supematant that was then added with anequivalent volume of 2×SDS sample buffer (containing 0.1%β-mercaptoethanol) to use as a test sample. In the meanwhile, the cellsin which the various types of HtrA2 were co-expressed with CREBL1 orATF6 were added with 200 μl of lysis buffer B and an equivalent volumeof 2×SDS sample buffer (containing 0.1% β-mercaptoethanol), and thensubjected to sonication followed by boiling to use as a test sample

Expression of SHC3 (p64), SHC (p52), ATF6, and CREBL1 and thedegradation of these proteins were detected by Western blotting using 10μl of the test sample thus obtained. Anti-c-Myc (9E10) antibody was usedas a primary antibody for detection, and horseradish peroxidase (HRP)conjugated anti-mouse IgG antibody (Cell Signaling) was used as asecondary antibody. Detection was carried out using ECL Western BlottingDetection System.

<Results>

In the analysis using cells in which the active mutant (mature HtrA2(ΔAVPS) or mature HtrA2 (GVPS)) was co-expressed with CREBL1, the bandof CREBL1 was significantly reduced (upper panel of FIG. 2-A). In theanalysis using cells in which the active mutant was co-expressed withATF6, the band of ATF6 was significantly reduced (upper panel of FIG.2-B). In the analysis using cells in which the active mutant wasco-expressed with SHC3 (p64), the band of SHC3 (p64) was significantlyreduced (upper panel of FIG. 2-C). In the analysis using cells in whichthe active mutant was co-expressed with SHC3 (p52), the band of SHC3(p52) was significantly reduced (upper panel of FIG. 2-D). On the otherhand, in the analysis using cells in which the inactive mutant (matureHtrA2 S306 (ΔAVPS) or mature HtrA2 S306 (GVPS)) was co-expressed withCREBL1, the reduction of the band of CREBL1 was not observed (upperpanel of FIG. 2-A). In the analysis using cells in which the inactivemutant was co-expressed with ATF6, the reduction of the band of ATF6 wasnot observed (upper panel of FIG. 2-B). In the analysis using cells inwhich the inactive mutant was co-expressed with SHC3 (p64), thereduction of the band of SHC3 (p64) was not observed (upper panel ofFIG. 2-C). In the analysis using cells in which the inactive mutant wasco-expressed with SHC3 (p52), the reduction of the band of SHC3 (p52)was not observed (upper panel of FIG. 2-D). The expression of each HtrA2mutant in the cells was almost same among the cells (lower panel of FIG.2-A, 2-B, 2-C and 2-D).

As mentioned above, it has been revealed that all of CREBL1, ATF6, SHC3(p64), and SHC3 (p52) are degraded in the cells by active mature HtrA2(ΔAVPS) or by active mature HtrA2 (GVPS). On the other hand, any ofCREBL1, ATF6, SHC3 (p64), and SHC3 (p52) is not degraded by inactivemature HtrA2 (S306A, ΔAVPS) or by inactive mature HtrA2 (S306A, GVPS).

EXAMPLE 4

(Investigation of Degradation Pattern by HtrA2)

Degradation pattern of CREBL1, SHC3 p64), or SHC3 (p52) by HtrA2 wasinvestigated. The investigation was performed by in vitro protease assaywith HtrA2 using CREBL1, SHC3 (p64), or SHC3 (p52) which is labeled withbiotin.

<Materials and Their Preparations>

1. Mature HtrA2 and mature HtrA2 (S306A)

Mature HtrA2 and mature HtrA2 (S306A) were prepared by similarprocedures as used in Example 2 for use.

2. animal cell expression plasmid for CREBL1, SHC3 p64) and SHC3 (p52)The animal cell expression plasmid for CREBL1 was prepared by digestingCREBL1, which had been cloned into pCR-BluntIl-TOPO vector by similarprocedures as used in Example 2, with BamHI and XhoI, and thenintegrated it into pcDNA3.1/His.

The animal cell expression plasmid for SHC3 (p64) was prepared bydigesting SHC3 (p64), which had been cloned into pCR4BIunt-TOPO vectorby similar procedures as used in Example 2, with EcoRI and XhoI, andthen integrated it into pcDNA3.1/V5-His.

The animal cell expression plasmid for SHC3 (p52) was prepared bydigesting SHC3 (p52), which had been cloned into pCMV-Tag5 by similarprocedures as used in Example 2, with PvuII and XhoI, and then ligatedit to pcDNA3.1/V5-His digested with EcoRV and XhoI.

3. Preparation of biotinylated CREBL1, biotinylated SHC3 (p64), andbiotinylated SHC3 (p52) using in vitro translation reaction system

The preparation of biotinylated CREBL1, biotinylated SHC3 (p64), andbiotinylated SHC3 (p52) was performed by in vitro translation reactionsystem (T_(N)T® Transcription/Translation System; Promega) using rabbitreticulocyte lysate.

More concretely, first 1.5 μl of the expression plasmid, 1 μl of 1 mMmethionine, 1 μl of biotinylated lysine tRNA (Promega), and 6.5 μl ofnuclease free water were added to 40 μl of in vitro translation reactionsolution (T_(N)T®Quick Master Mix) to obtain total volume of 50 μl, andreacted for at 30° C. for 1.5 hours. Following this, 12.5 μl of 50 μlreaction solution was sampled, followed by adding with 2×SDS samplebuffer and boiling, to detect the expression of biotinylated CREBL1,biotinylated SHC3 p64), and biotinylated SHC3 (p52) by Western blotting.The detection was carried out using streptavidin HRP (Promega) andTranscend™ Chemiluminescent substrate (Tranend™ Non-RadioactiveTranslation detection system; Promega). As a result, biotinylation ofthese proteins was confirmed.

<Methods>

The reaction solution was used as the sample for protease assay. 12.5 μleach of the reaction solution was dispensed to three tubes (Nos. 2 to4). 25 μl of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.01% TritonX-100 wasadded asthe control to tubeNo.2, 25 μl of 200 μg/ml mature HtrA2solution was added to tube No.3, and 25 μl of 200 μg/ml mature HtrA2(S306A) solution was added to tube No.4, respectively, and mixed. Thesolution in each tube (Nos. 2 to 4) was further divided into two tubesso as to the tubes include an equivalent volume of solution, while onetube was subjected to reaction at 37° C. for four hours and the othertube was subjected to reaction at 370C overnight. After the reaction,2×SDS sample buffer was added to each of tubes, and subsequentlysubjected to boiling, to detect the degradation pattern of biotinylatedCREBL1, biotinylated SHC3 (p64), and biotinylated SHC3 (p52) by Westernblotting.

The detection of the degradation was carried out using streptavidin HRPand Trascend™ Chemiluminescent substrate while biotinylated lysineresidue in each protein was used as the index.

Further, in orderto determine the degradation site, the membrane usedfor Westem blotting was subjected to treatment for removing thestreptavidin HRP, and then used for detecting the degradation pattern ofbiotinylated CREBL1, biotinylated SHC3 (p64), and biotinylated SHC3(p52) using an antibody against the tag ligated to each protein. Anti-V5antibody (Invitrogen) and HRP labeled anti-mouse IgG antibody (CellSignaling) were used as a primary antibody and a secondary antibody,respectively, for detecting SHC3. Anti-Xpress antibody (Invitrogen) andHRP labeled anti-mouse IgG antibody (Cell Signaling) were used as aprimary antibody and a secondary antibody, respectively, for detectingCREBL1. The detection of degradation was carried out using ECL WesternBlotting Detection Reagents (Amersham).

<Results>

Results of the study for the degradation pattern of CREBL1, SHC3 (p64),or SHC3 (p52) by mature HtrA2 using biotinylated lysine residue in theprotein as an index are shown in FIG. 3-A, FIG. 3-B nd FIG. 3-C,respectively. Similarly, results of the study for the degradationpattern of CREBL1, SHC3 (p64), or SHC3 (p52) using a tag ligated to theN-terninal or C-teminal of the protein as an index are shown in FIG.4-A, FIG. 4-B and FIG. 4-C, respectively.

In the study for the degradation pattern using biotinylated lysineresidue as an index, remarkable reduction of the band of biotinylatedCREBL1 was observed, and further, the band that was supposed to be adegradation product of CREBL1 was detected around 50 kDa (FIG. 3-A).However, in the study for the degradation pattem using the anti-tagantibody, the band that was supposed to be a degradation product ofCREBL1 which was detected around 50 kDa in FIG. 3-A could not bedetected. Moreover, any band indicating the degradation product ofCREBL1 with different size was not detected (FIG. 4-A). From thisfinding, it is believed that CREBL1 was degraded by HtrA2 at severalsites.

In the study for the degradation pattern using biotinylated lysineresidue as an index, remarkable reduction of the band of biotinylatedSHC3 (p64) was observed, and further, the bands that were supposed to bea degradation products of SHC3 (p64) were detected around 70 kDa, 40kDa, and 30 kDa (FIG. 3-B). In addition, the same bands were detected inthe study for the degradation pattern using the anti-tag antibody (FIG.4-B). From this finding, it can be considered that SHC3 (p64) may bedegraded by mature HtrA2 at several sites.

In the study for the degradation pattern using biotinylated lysineresidue as an index, remarkable reduction of the band of biotinylatedSHC3 (p52) was observed, but the band that was supposed to be adegradation product of SHC3 (p52) was not detected (FIG. 3-C). However,in the study for the degradation pattern using the anti-tag antibody,the band that was supposed to be a degradation products of SHC3 (p52)was detected around 4OkDa (FIG. 4-C). From this finding, it is believedthat SHC3 (p52) was degraded by HtrA2 at several sites.

As described above, the degradation of the aforementioned protein byHtrA2 was detected by detecting a labeled chemical that was used forlabeling the inside of the protein. From this finding, it has beenrevealed that the degradation of the protein by HtrA2 identified bydetection of the tag ligated to the N-terminal of each protein (Examples2 and 3) is not an apparent degradation due to the cleavage of the tag,but is a degradation resulting from the action of HtrA2 on the inside ofeach protein.

INDUSTRIAL APPLICABILITY

The present invention can be utilized in inhibition of cell death, suchas neural cell death attributable to the degradation by HtrA2 of atleast one of SHC3, ATF6 and CREBL1, and in prevention, treatment, orcontrol of the diseases accompanied with neural cell death, such asneurodegenerative diseases, attributable to the same. Thus, the presentinvention is extremely useful in the pharmaceutical fields. Further, thepresent invention can be utilized in the research field, such as theinvestigation of cell death caused by HtrA2, for example, cell death dueto the endoplasmic reticulum stress, or neural cell death.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: DNA encoding HtrA2 precursor protein

-   SEQ ID NO: 2: HtrA2 precursor protein-   SEQ ID NO: 3: DNA encoding mature HtrA2-   SEQ ID NO: 4: mature HtrA2-   SEQ ID NO: 5: Polynucleotide consisting of the same nucleotide    sequence of SEQ ID NO: 3, wherein the nucleotide of position 520 is    g; DNA encoding mature HtrA2 (S306A)-   SEQ ID NO: 6: Polypeptide consisting of the same amino acid sequence    of SEQ ID NO: 4, wherein the 174th amino acid residue is substituted    by Ala; mature HtrA2 (S306A)-   SEQ ID NO: 7: Polynucleotide consisting of the same nucleotide    sequence of SEQ ID NO: 3, wherein the nucleotides of position 4-15    are deleted; DNA encoding mature HtrA2 (delta AVPS)-   SEQ ID NO: 8: Polypeptide consisting of the same amino acid sequence    of SEQ ID NO: 4, wherein the amino acid residues from the 2nd to the    5th are deleted; mature HtrA2 (delta AVPS)-   SEQ ID NO: 9: Polynucleotide consisting of the same nucleotide    sequence of SEQ ID NO: 3, wherein the nucleotide of position 5 is g;    DNA encoding mature HtrA2 (GVPS)-   SEQ ID NO: 10: Polypeptide consisting of the same amino acid    sequence of SEQ ID NO: 4, wherein the 2nd amino acid residue is    substituted by Gly; mature HtrA2 (GVPS)-   SEQ ID NO: 11: Polynucleotide consisting of the same nucleotide    sequence of SEQ ID NO: 5, wherein the nucleotides of position 4-15    are deleted; DNA encoding mature HtrA2 (S306A, delta AVPS)-   SEQ ID NO: 12: Polypeptide consisting of the same amino acid    sequence of SEQ ID NO: 6, wherein the amino acid residues from the    2nd to the 5th are deleted; mature HtrA2 (S306A, delta AVPS)-   SEQ ID NO: 13: Polynucleotide consisting of the same nucleotide    sequence of SEQ ID NO: 5, wherein the nucleotide of position 5 is g;    DNA encoding mature HtrA2 (S306A, GVPS)-   SEQ ID NO: 14: Polypeptide consisting of the same amino acid    sequence of SEQ ID NO: 6, wherein the 2nd amino acid residue is    substituted by Gly; mature HtrA2 (S306A, GVPS)-   SEQ ID NO: 15: DNA encoding SHC3-   SEQ ID NO: 16: SHC3-   SEQ ID NO: 17: DNA encoding CREBL1-   SEQ IDNO: 18: CREBL1-   SEQ ID NO: 19: DNA encoding ATF6-   SEQ ID NO: 20: ATF6-   SEQ ID NO: 21: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 for use as a primer to obtain mature HtrA2    DNA-   SEQ ID NO: 22: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 for use as a primer to obtain mature HtrA2    DNA-   SEQ ID NO: 23: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 foruse as a primer to obtain mature HtrA2    (S306A) DNA-   SEQ ID NO: 24: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 for use as a primer to obtain mature HtrA2    (S306A) DNA-   SEQ ID NO: 25: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 15 for use as a primer to obtain SHC3 DNA-   SEQ ID NO: 26: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 15 for use as a primer to obtain SHC3 DNA-   SEQ ID NO: 27: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 17 for use as a primer to obtain CREBL1 DNA-   SEQ ID NO: 28: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 17 for use as a primer to obtain CREBL1 DNA-   SEQ ID NO: 29: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 17 for use as a primer to obtain CREBL1 DNA-   SEQ ID NO: 30: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 17 for use as a primer to obtain CREBL1 DNA-   SEQ ID NO: 31: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 19 for use as a primer to obtain ATF6 DNA-   SEQ ID NO: 32: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 19 for use as a primer to obtain ATF6 DNA-   SEQ ID NO: 33: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 19 for use as a primer to obtain ATF6 DNA-   SEQ ID NO: 34: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 19 for use as a primer to obtain ATF6 DNA-   SEQ ID NO: 35: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 19 for use as a primer to obtain ATF6 DNA-   SEQ ID NO: 36: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 for use as a primer to obtain mature HtrA2    (delta AVPS) DNA-   SEQ ID NO: 37: Designed polynucleotide based on the nucleotide    sequence of SEQ ID NO: 3 for use as a primer to obtain mature HtrA2    (GVPS) DNA

1. A method for inhibiting neural cell death, comprising inhibiting thedegradation by HtrA2 (high temperature requirement protein A2) of atleast one of SHC3 (src homology 2 domain containing transforming proteinC3), ATF6 (activating transcription factor 6), or CREBL1 (cAMPresponsive element binding protein-like 1).
 2. A method for inhibitingneural cell death of claim 1, comprising using one or more compoundsthat inhibit the degradation by HtrA2 of at least one of SHC3, ATF6 orCREBL
 1. 3. The method for inhibiting neural cell death according toclaim 1 , in which the neural cell death is neural cell deathattributable to brain ischemia.
 4. A method for preventing, treating orcontrolling brain ischemia or neurodegenerative disease, comprisinginhibiting the degradation by HtrA2 of at least one of SHC3, ATF6 orCREBL1.
 5. The method according to claim 4, in which theneurodegenerative disease is Alzheimer's disease, Parkinson's disease,polyglutamine disease, prion disease, or amyotrophic lateral sclerosis.6. A method of identifying an agent of claim 8, that inhibits thedegradation by HtrA2 of at least one of SHC3, ATF6 or CREBL1, comprisingcontacting HtrA2 and at least one of SHC3, ATF6 or CREBL1 with acompound under conditions that allow the degradation by HtrA2 of atleast one of SHC3, ATF6 or CREBL1; introducing a system using a signaland a marker capable of detecting at least one of SHC3, ATF6 or CREBL1;detecting the presence or absence and/or change of the signal and themarker; and determining whether the compound inhibits the degradation ofat least one of SHC3, ATF6 or CREBL1.
 7. A method of identifying anagent of claim 8 that inhibits the degradation by HtrA2 of at least oneof SHC3, ATF6 or CREBL1, comprising contacting HtrA2 and at least one ofSHC3, ATF6 or CREBL1 with a compound under conditions that allow thedegradation by HtrA2 of at least one of SHC3, ATF6 or CREBL1; detectingthe presence or absence of at least one of SHC3, ATF6 or CREBL1, and/ormeasuring the change of the amount thereof; or detecting the presence orabsence of the degradation product of at least one of SHC3, ATF6 orCREBL1, and/or measuring the change of the amount thereof; anddetermining whether the compound inhibits the degradation of at leastone of SHC3, ATF6 and CREBL1.
 8. An agent for inhibiting neural celldeath or for preventing treating or controlling brain ischemia orneurodegenerative disease, comprising one or more compounds that inhibitthe degradation by HtrA2 of at least one of SHC3, ATF6 or CREBL1.
 9. Theagent for inhibiting neural cell death according to claim 8, wherein theneural cell death is neural cell death attributable to brain ischemia.10. (canceled)
 11. The agent for preventing, treating or controllingneurodegenerative disease according to claim 8, wherein theneurodegenerative disease is Alzheimer's disease, Parkinson's disease,polyglutamine disease, prion disease, or amyotrophic lateral sclerosis.12. A reagent kit, comprising at least one selected from the groupconsisting of HtrA2, a polynucleotide encoding HtrA2, and a vectorcontaining the polynucleotide encoding HtrA2; and at least one selectedfrom the group consisting of SHC3, ATF6, CREBL1, a polynucleotideencoding at least one of SHC3, ATF6 and CREBL1, and a vector containingthe polynucleotide encoding at least one of SHC3, ATF6 and CREBL1. 13.The method for inhibiting neural cell death according to claim 2, inwhich the neural cell death is neural cell death attributable to brainischemia.